Specific single-cell isolation and genomic amplification of uncultured microorganisms

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.     

New strategies for cultivation and detection of previously uncultured microbes

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O(2) [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO(2) (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO(2). A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.     

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions

Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6 months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the Deep-Sea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.     

Effect of media composition,including gelling agents,on isolation of previously uncultured rumen bacteria

The aim of this study was to develop novel anaerobic media using gellan gum for the isolation of previously uncultured rumen bacteria. Four anaerobic media, a basal liquid medium (BM) with agar (A‐BM), a modified BM (MBM) with agar (A‐MBM), an MBM with phytagel (P‐MBM) and an MBM with gelrite (G‐MBM) were used for the isolation of rumen bacteria and evaluated for the growth of previously uncultured rumen bacteria. Of the 214 isolates composed of 144 OTUs, 103 isolates (83 OTUs) were previously uncultured rumen bacteria. Most of the previously uncultured strains were obtained from A‐MBM, G‐MBM and P‐MBM, but the predominant cultural members, isolated from each medium, differed. A‐MBM and G‐MBM showed significantly higher numbers of different OTUs derived from isolates than A‐BM (P < 0·05). The Shannon index indicated that the isolates of A‐MBM showed the highest diversity (H′ = 3·89) compared with those of G‐MBM, P‐MBM and A‐BM (H′ = 3·59, 3·23 and 3·39, respectively). Although previously uncultured rumen bacteria were isolated from all media used, the ratio of previously uncultured bacteria to total isolates was increased in A‐MBM, P‐MBM and G‐MBM.     

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.     

Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes

We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes) . Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes.     

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 microg ml(-1). The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as 'Candidatus Nitrospira defluvii'.     

Macrophyte species drive the variation of bacterioplankton community composition in a shallow freshwater lake

Macrophytes play an important role in structuring aquatic ecosystems. In this study, we explored whether macrophyte species are involved in determining the bacterioplankton community composition (BCC) in shallow freshwater lakes. The BCC in field areas dominated by different macrophyte species in Taihu Lake, a large, shallow freshwater lake, was investigated over a 1-year period. Subsequently, microcosm experiments were conducted to determine if single species of different types of macrophytes in an isolated environment would alter the BCC. Denaturing gradient gel electrophoresis (DGGE), followed by cloning and sequence analysis of selected samples, was employed to analyze the BCC. The DGGE results of the field investigations indicated that the BCC changed significantly from season to season and that the presence of different macrophyte species resulted in lower BCC similarities in the summer and fall. LIBSHUFF analysis of selected clone libraries from the summer demonstrated different BCCs in the water column surrounding different macrophytes. Relative to the field observations, the microcosm studies indicated that the BCC differed more pronouncedly when associated with different species of macrophytes, which was also supported by LIBSHUFF analysis of the selected clone libraries. Overall, this study suggested that macrophyte species might be an important factor in determining the composition of bacterial communities in this shallow freshwater lake and that the species-specific influence of macrophytes on BCC is variable with the season and distance.     

The structure and stability of the bacterioplankton community in Antarctic freshwater lakes, subject to extremely rapid environmental change

In this study, variation in the bacterioplankton community structure of three Antarctic lakes of different nutrient status, was determined in relation to physical and chemical gradients at depth and at time intervals, across the seasonal transition from winter ice-cover to the summer ice-free period. The three lakes studied were: Moss Lake (low nutrient, with typical nutrient concentrations of 80 microg l(-1) nitrate and 10 microg l(-1) dissolved reactive phosphate), Sombre Lake (low nutrient, but becoming progressively enriched, with typical nutrient concentrations of 185 microg l(-1) nitrate and 7 microg l(-1) dissolved reactive phosphate) and Heywood Lake (enriched, with typical nutrient concentrations of 1180 microg l(-1) nitrate and 124 microg l(-1) dissolved reactive phosphate). Bacterioplankton community structure was determined using a combination of PCR amplification of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE). Results indicated marked changes in this bacterioplankton community structure, which were particularly associated with the transition period. However, significant changes also occurred during the period of holomixis. Comparison of the results from lakes of different nutrient status suggest that increased levels of nutrient input, and in the timing and duration of ice cover will lead to marked changes in the structure and stability of the bacterioplankton community at existing levels of environmental change.     

Grazer and virus-induced mortality of bacterioplankton accelerates development of Flectobacillus populations in a freshwater community

We present a detailed analysis of the effects of distinct bacterial mortality factors, viral lysis and heterotrophic nanoflagellates (HNF) bacterivory, associated with the development of filamentous Flectobacillus populations. Reservoir bacterioplankton communities were subjected to additions of both HNF and viruses together, or HNF alone, and then incubated in situ in dialyses bags. For distinct bacterial groups, mortality or growth stimulation was analysed by examining bacterial prey ingested in HNF food vacuoles with fluorescence in situ hybridization (FISH) and via FISH with microautoradiography (MAR-FISH). We also developed a semi-quantitative MAR-FISH-based estimation of relative activities of Flectobacillus populations (targeted by the R-FL615 probe). Bacterial groups vulnerable to HNF predation (mainly clusters of Betaproteobacteria), or discriminated against (Actinobacteria), were detected. Bacterial lineages most vulnerable to virus-lysis (mainly the Betaproteobacteria not targeted by the R-BT065 probe, of the Polynucleobacter cluster) were identified by comparing treatments with HNF alone to HNF and viruses together. Filaments affiliated with the Flectobacillus cluster appeared in both treatments, but were about twice as abundant, long and active as in incubations with viruses and HNF as compared with HNF alone. Viruses appeared to selectively suppress several bacterial groups, perhaps enhancing substrate availability thus stimulating growth and activity of filamentous Flectobacillus.     

Links between resources, C metabolism and the major components of bacterioplankton community structure across a range of freshwater ecosystems

We explored the patterns in bacterioplankton community metabolism (BCM) and four components of community structure [composition (BCC), metabolic capacities (MC), physiological structure (PS) and single-cell characteristics (SCC)], between lakes, rivers and marshes within a watershed in Québec, to assess the connections that exist between them and with the main resources (organic matter, nutrients). Habitat types were well segregated by both resources and BCM and their corresponding dissimilarity matrices were significantly correlated, suggesting that BCM tracks resource conditions in a consistent manner across ecosystem types. MC also segregated the various habitats and was correlated to BCM but less so to resources, whereas BCC at times resulted in a clear separation of habitats, but was rarely correlated to resources and never to BCM, suggesting a higher degree of ecosystem specificity at this particular level. Finally, there was no clear separation of habitats in terms of PS and SCC, and none covaried with resources or BCM. The habitat patterns based on these different components of structure were rarely correlated to each other, indicating weak deterministic connections between them. MC appears to mediate the link between resources and BCM more directly and consistently across systems; BCC appears to be more influenced by ecosystem-specific factors that weaken its overall connection to both resources and BCM, whereas PS and SCC show no discernible patterns. Our results thus suggest that the bottom-up regulation of BCM by resources is mediated by complex shifts within components of community structure that can be directional, ecosystem-specific or apparently random, which combined nevertheless result in a systematic overall response to resources in terms of C metabolism.     

Laboratory cultivation of widespread and previously uncultured soil bacteria

Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum ACTINOBACTERIA: In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.     

Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems

In order to extend previous comparisons between coastal marine bacterioplankton communities and their open ocean and freshwater counterparts, here we summarize and provide new data on a clone library of 105 SSU rRNA genes recovered from seawater collected over the western continental shelf of the USA in the Pacific Ocean. Comparisons to previously published data revealed that this coastal bacterioplankton clone library was dominated by SSU rRNA gene phylotypes originally described from surface waters of the open ocean, but also revealed unique SSU rRNA gene lineages of beta Proteobacteria related to those found in clone libraries from freshwater habitats. beta Proteobacteria lineages common to coastal and freshwater samples included members of a clade of obligately methylotrophic bacteria, SSU rRNA genes affiliated with Xylophilus ampelinus, and a clade related to the genus Duganella. In addition, SSU rRNA genes were recovered from such previously recognized marine bacterioplankton SSU rRNA gene clone clusters as the SAR86, SAR11, and SAR116 clusters within the class Proteobacteria, the Roseobacter clade of the alpha subclass of the Proteobacteria, the marine group A/SAR406 cluster, and the marine Actinobacteria clade. Overall, these results support and extend previous observations concerning the global distribution of several marine planktonic prokaryote SSU rRNA gene phylotypes, but also show that coastal bacterioplankton communities contain SSU rRNA gene lineages (and presumably bacterioplankton) shown previously to be prevalent in freshwater habitats.     

Effective isolation of bacterioplankton genus Polynucleobacter from freshwater environments grown on photochemically degraded dissolved organic matter

Effective isolation of freshwater bacterioplankton belonging to genus Polynucleobacter from a shallow eutrophic lake and its tributary was achieved by size-selective filtration with a 0.7-μm pore filter and cultivation on R2A agar medium. Partial 16S rRNA gene analysis showed that over 80% of all the strains were highly similar to the Polynucleobacter cluster. Essential medium components for effective cultivation are pyruvate, yeast extract and peptone, whereas soluble starch and glucose are not necessary. Isolate KF001 (affiliated with Polynucleobacter subcluster D) has a strict requirement for organic acids as carbon sources, and we hypothesize that the Polynucleobacter cluster of bacteria could utilize compounds formed via photochemically dissolved organic matter (DOM) degradation for growth. Because organic acids form from solar radiation of DOM in aquatic environments, carbon sources that are typical products of DOM photochemical degradation were added to the medium. These compounds were readily utilized by KF001 in this study. Finally, we observed the stimulation of strain KF001 activity by photochemical degradation of natural lake water. Our findings suggest a carbon flow of DOM photoproducts to Polynucleobacter in the freshwater microbial loop.     

Bio-optical characterization of selected cyanobacteria strains present in marine and freshwater ecosystems

The optical properties, i.e., absorption and scattering spectra of ten strains of cyanobacteria from the Baltic Sea and Pomeranian lakes (Aphanizomenon flos-aquae KAC 15, Microcystis aeruginosa CCNP 1101, Anabaena sp. CCNP 1406, Synechocystis salina CCNP 1104, Phormidium sp. CCNP 1317, Nodularia spumigena CCNP 1401, Synechococcus sp. CCNP 1108, Nostoc sp. CCNP 1411, Cyanobacterium sp. CCNP 1105, Pseudanabaena cf. galeata CCNP 1312) grown under low light conditions were investigated. Moreover, the chlorophylls, carotenoids, and phycobilin composition as well as the size structure of chosen cyanobacteria were measured. Studied species revealed high diversity both in optical properties with the absorption spectra similarity index ranging from 0.67 to 0.94 and the pigment composition. The chlorophyll-specific absorption coefficient at 440 nm a _ph *(440) varied between 0.017 and 0.065 m² mg^?1. The influence of the package effect was only observed in the case of large filamentous cyanobacteria like N. spumigena or Nostoc sp. Interestingly, the package effect factor Q _a *(675) for large-celled Anabaena sp. was 0.92. Besides chlorophyll a, only echinenone, β-carotene, and phycocyanin were present in all analyzed cyanobacteria strains. Zeaxanthin, which is widely used as a marker pigment for cyanobacteria, was absent in the toxic N. spumigena and Anabaena sp., which are the species that occur in the Baltic Sea most frequently causing summer cyanobacterial blooms. The investigation also showed that the sample preservation technique can introduce some major errors within the absorption band affected by the phycocyanin absorption.     

Placental anticoagulant proteins: isolation and comparative characterization four members of the lipocortin family

Previously we isolated and characterized a placental anticoagulant protein (PAP or PAP-I), which is a Ca2+-dependent phospholipid binding protein [Funakoshi et al. (1987) Biochemistry 26, 5572] and a member of the lipocortin family [Funakoshi et al. (1987) Biochemistry 26, 8087]. In this study, three additional anticoagulant proteins (PAP-II, PAP-III, and PAP-IV) were simultaneously isolated from human placental homogenates prepared in the presence of 5 mM ethylenediaminetetraacetic acid. The isoelectric points of PAP-I, PAP-II, PAP-III, and PAP-IV were 4.8, 6.1, 5.9, and 8.1, respectively, and their apparent molecular weights were 32,000, 33,000, 34,000, and 34,500, respectively. Amino acid sequences of cyanogen bromide fragments of these proteins showed that PAP-III was a previously unrecognized member of the lipocortin family, while PAP-II was probably the human homologue of porcine protein II and PAP-IV was a derivative of lipocortin II truncated near the amino terminus. Comparative studies showed that all four proteins inhibited blood clotting and phospholipase A2 activity with potencies consistent with their measured relative affinities for anionic phospholipid vesicles. However, PAP-IV bound to phospholipid vesicles approximately 160-fold more weakly than PAP-I, while PAP-II and PAP-III bound only 2-fold and 3-fold more weakly. These results increase to six the number of lipocortin-like proteins known to exist in human placenta. The observed differences in phospholipid binding may indicate functional differences among the members of the lipocortin family despite their considerable structural similarities.     

Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of Geothermobacterium ferrireducens gen. nov., sp. nov

It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 microm by 1.0 to 1.2 microm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100 degrees C with an optimum of 85 to 90 degrees C. To our knowledge this is the highest temperature optimum of any organism in the BACTERIA: Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).     

Rapid isolation and characterization of hybridization selected recombinants from lambda genomic libraries

This paper describes an efficient protocol for the screening of lambda genomic libraries, plaque and DNA purification, and probe characterization by a combination of new and recently described techniques. The protocol has allowed large numbers of human subchromosome-specific probes to be rapidly generated from an EMBL3 library of human-mouse somatic cell hybrid DNA. The protocol affords considerable savings in time and effort over previous procedures.